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State-dependent inactivation of the alpha1G T-type calcium channel.

Serrano JR, Perez-Reyes E, Jones SW - J. Gen. Physiol. (1999)

Bottom Line: Recovery was similar after 60-ms steps to -20 mV or 600-ms steps to -70 mV, suggesting rapid equilibration of open- and closed-state inactivation.The results are well described by a kinetic model where inactivation is allosterically coupled to the movement of the first three voltage sensors to activate.One consequence of state-dependent inactivation is that alpha1G channels continue to inactivate after repolarization, primarily from the open state, which leads to cumulative inactivation during repetitive pulses.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology and Biophysics, Case Western Reserve University, Cleveland, Ohio 44106, USA.

ABSTRACT
We have examined the kinetics of whole-cell T-current in HEK 293 cells stably expressing the alpha1G channel, with symmetrical Na(+)(i) and Na(+)(o) and 2 mM Ca(2+)(o). After brief strong depolarization to activate the channels (2 ms at +60 mV; holding potential -100 mV), currents relaxed exponentially at all voltages. The time constant of the relaxation was exponentially voltage dependent from -120 to -70 mV (e-fold for 31 mV; tau = 2.5 ms at -100 mV), but tau = 12-17 ms from-40 to +60 mV. This suggests a mixture of voltage-dependent deactivation (dominating at very negative voltages) and nearly voltage-independent inactivation. Inactivation measured by test pulses following that protocol was consistent with open-state inactivation. During depolarizations lasting 100-300 ms, inactivation was strong but incomplete (approximately 98%). Inactivation was also produced by long, weak depolarizations (tau = 220 ms at -80 mV; V(1/2) = -82 mV), which could not be explained by voltage-independent inactivation exclusively from the open state. Recovery from inactivation was exponential and fast (tau = 85 ms at -100 mV), but weakly voltage dependent. Recovery was similar after 60-ms steps to -20 mV or 600-ms steps to -70 mV, suggesting rapid equilibration of open- and closed-state inactivation. There was little current at -100 mV during recovery from inactivation, consistent with

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Nonmonotonic recovery from inactivation. Three pulses were given (each 5 ms to −20 mV), with a variable interval (1–300 ms) between the first two pulses (P1 and P2), and 20 ms between P2 and P3. (A) Sample records for P1–P2 intervals of 1 and 10 ms. Note that the 1-ms interval at −100 mV is not long enough for many channels to close. The P2 current is clearly larger for the 1-ms interval, and the P3 current is slightly larger (easiest to see in the tail current amplitudes following P3, see dashed lines). Cell a8702, 3 kHz Gaussian filter. (B) The time course of recovery from inactivation, averaged from seven cells. Note the log time scale. The decrease in P2 amplitude from 1 to 10 ms could reflect either continued inactivation during the tail current, or residual activation remaining from P1 (see A). But the 20-ms P2–P3 interval is long enough for channels to fully deactivate, so the decrease in P3 amplitude from 1 to 10 ms indicates a genuinely nonmonotonic time course for recovery from inactivation.
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Figure 13: Nonmonotonic recovery from inactivation. Three pulses were given (each 5 ms to −20 mV), with a variable interval (1–300 ms) between the first two pulses (P1 and P2), and 20 ms between P2 and P3. (A) Sample records for P1–P2 intervals of 1 and 10 ms. Note that the 1-ms interval at −100 mV is not long enough for many channels to close. The P2 current is clearly larger for the 1-ms interval, and the P3 current is slightly larger (easiest to see in the tail current amplitudes following P3, see dashed lines). Cell a8702, 3 kHz Gaussian filter. (B) The time course of recovery from inactivation, averaged from seven cells. Note the log time scale. The decrease in P2 amplitude from 1 to 10 ms could reflect either continued inactivation during the tail current, or residual activation remaining from P1 (see A). But the 20-ms P2–P3 interval is long enough for channels to fully deactivate, so the decrease in P3 amplitude from 1 to 10 ms indicates a genuinely nonmonotonic time course for recovery from inactivation.

Mentions: Another sign of state-dependent inactivation is “nonmonotonic recovery from inactivation” (Neher and Lux 1971; Marom and Levitan 1994). For pairs of brief depolarizations, channels continue to inactivate during the initial part of the interpulse interval, before recovery from inactivation begins, producing a U-shaped time course for the current measured during the second pulse (Fig. 13). However, apparent nonmonotonic recovery can be observed for interpulse intervals that are not long enough to fully close the channel, if that leads to more channel activation during the second pulse (Gillespie and Meves 1980). That is, a larger test pulse current could result from greater channel activation, rather than less inactivation, for very brief intervals. To exclude this possibility, we delivered a third pulse, after allowing 20 ms for complete channel closing. Currents during the third pulse also showed a U-shaped time dependence (Fig. 13), although inactivation during the 20-ms tail current (and at early times during the third pulse) made the U shape less dramatic. Since nonmonotonic recovery would not occur at all if inactivation were strictly voltage dependent, this is good evidence for state-dependent inactivation.


State-dependent inactivation of the alpha1G T-type calcium channel.

Serrano JR, Perez-Reyes E, Jones SW - J. Gen. Physiol. (1999)

Nonmonotonic recovery from inactivation. Three pulses were given (each 5 ms to −20 mV), with a variable interval (1–300 ms) between the first two pulses (P1 and P2), and 20 ms between P2 and P3. (A) Sample records for P1–P2 intervals of 1 and 10 ms. Note that the 1-ms interval at −100 mV is not long enough for many channels to close. The P2 current is clearly larger for the 1-ms interval, and the P3 current is slightly larger (easiest to see in the tail current amplitudes following P3, see dashed lines). Cell a8702, 3 kHz Gaussian filter. (B) The time course of recovery from inactivation, averaged from seven cells. Note the log time scale. The decrease in P2 amplitude from 1 to 10 ms could reflect either continued inactivation during the tail current, or residual activation remaining from P1 (see A). But the 20-ms P2–P3 interval is long enough for channels to fully deactivate, so the decrease in P3 amplitude from 1 to 10 ms indicates a genuinely nonmonotonic time course for recovery from inactivation.
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Figure 13: Nonmonotonic recovery from inactivation. Three pulses were given (each 5 ms to −20 mV), with a variable interval (1–300 ms) between the first two pulses (P1 and P2), and 20 ms between P2 and P3. (A) Sample records for P1–P2 intervals of 1 and 10 ms. Note that the 1-ms interval at −100 mV is not long enough for many channels to close. The P2 current is clearly larger for the 1-ms interval, and the P3 current is slightly larger (easiest to see in the tail current amplitudes following P3, see dashed lines). Cell a8702, 3 kHz Gaussian filter. (B) The time course of recovery from inactivation, averaged from seven cells. Note the log time scale. The decrease in P2 amplitude from 1 to 10 ms could reflect either continued inactivation during the tail current, or residual activation remaining from P1 (see A). But the 20-ms P2–P3 interval is long enough for channels to fully deactivate, so the decrease in P3 amplitude from 1 to 10 ms indicates a genuinely nonmonotonic time course for recovery from inactivation.
Mentions: Another sign of state-dependent inactivation is “nonmonotonic recovery from inactivation” (Neher and Lux 1971; Marom and Levitan 1994). For pairs of brief depolarizations, channels continue to inactivate during the initial part of the interpulse interval, before recovery from inactivation begins, producing a U-shaped time course for the current measured during the second pulse (Fig. 13). However, apparent nonmonotonic recovery can be observed for interpulse intervals that are not long enough to fully close the channel, if that leads to more channel activation during the second pulse (Gillespie and Meves 1980). That is, a larger test pulse current could result from greater channel activation, rather than less inactivation, for very brief intervals. To exclude this possibility, we delivered a third pulse, after allowing 20 ms for complete channel closing. Currents during the third pulse also showed a U-shaped time dependence (Fig. 13), although inactivation during the 20-ms tail current (and at early times during the third pulse) made the U shape less dramatic. Since nonmonotonic recovery would not occur at all if inactivation were strictly voltage dependent, this is good evidence for state-dependent inactivation.

Bottom Line: Recovery was similar after 60-ms steps to -20 mV or 600-ms steps to -70 mV, suggesting rapid equilibration of open- and closed-state inactivation.The results are well described by a kinetic model where inactivation is allosterically coupled to the movement of the first three voltage sensors to activate.One consequence of state-dependent inactivation is that alpha1G channels continue to inactivate after repolarization, primarily from the open state, which leads to cumulative inactivation during repetitive pulses.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology and Biophysics, Case Western Reserve University, Cleveland, Ohio 44106, USA.

ABSTRACT
We have examined the kinetics of whole-cell T-current in HEK 293 cells stably expressing the alpha1G channel, with symmetrical Na(+)(i) and Na(+)(o) and 2 mM Ca(2+)(o). After brief strong depolarization to activate the channels (2 ms at +60 mV; holding potential -100 mV), currents relaxed exponentially at all voltages. The time constant of the relaxation was exponentially voltage dependent from -120 to -70 mV (e-fold for 31 mV; tau = 2.5 ms at -100 mV), but tau = 12-17 ms from-40 to +60 mV. This suggests a mixture of voltage-dependent deactivation (dominating at very negative voltages) and nearly voltage-independent inactivation. Inactivation measured by test pulses following that protocol was consistent with open-state inactivation. During depolarizations lasting 100-300 ms, inactivation was strong but incomplete (approximately 98%). Inactivation was also produced by long, weak depolarizations (tau = 220 ms at -80 mV; V(1/2) = -82 mV), which could not be explained by voltage-independent inactivation exclusively from the open state. Recovery from inactivation was exponential and fast (tau = 85 ms at -100 mV), but weakly voltage dependent. Recovery was similar after 60-ms steps to -20 mV or 600-ms steps to -70 mV, suggesting rapid equilibration of open- and closed-state inactivation. There was little current at -100 mV during recovery from inactivation, consistent with

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Related in: MedlinePlus